Islet amyloid polypeptide in insulinoma and in the islets of the pancreas of non-diabetic and diabetic subjects

Summary Amyloid deposition is a common pathological feature in insulinoma and in the islets of the pancreas in type-2 diabetic patients. The present immunohisto-chemical study revealed that normal B-cells, insulinoma, and amyloid deposits in insulinoma and diabetic pancreatic islets were commonly immunoreactive with antiserum to C-terminal synthetic tetradecapeptide of human islet amyloid polypeptide (IAPP) (24–37). Amyloid fibrils in insulinoma were also positive to IAPP by immunoelectron microscopy. A high level of IAPP was detected in the plasma and tissue of a insulinoma patient by radioimmunoassay suggesting that amyloid deposition in insulinoma is due to overproduction of IAPP. Amyloid deposits immunoreactive to IAPP were also seen in all diabetic pancreatic islets, but in no non-diabetic islets. There was much amyloid deposition in the islets of severe diabetics, whose B-cells demonstrated decreased immunoreactivities for IAPP and insulin. The IAPP content of the pancreas was 649.0 and 847.7 pg/mg wet weight in each of two diabetic patients, and 1034.6 and 1447.7 pg/mg wet weight in two non-diabetic patients. The present study revealed that IAPP is a bioactive peptide secreted from islet B-cells and are amyloidogenic peptide concerned in diabetogenensis and/or the progression of type-2 diabetes mellitus.     

IAPP as a regulator of glucose homeostasis and pancreatic hormone secretion (review).

IAPP is a 37-amino acid peptide that is predominantly expressed in pancreatic beta cells. Despite co-secretion from islets the relative amounts of IAPP and insulin may vary. Since IAPP was first described as the major peptide constituent of amyloid in the islets of Langerhans of subjects with type 2 diabetes and insulinoma, many studies have been devoted to investigating the role of IAPP in formation of amyloid deposits and in diabetes pathogenesis. However, there is growing evidence for IAPP as an active islet hormone in addition to insulin and glucagon in glucose metabolic control. An inhibitory effect is seen by IAPP on gastric emptying, glycogen synthesis in skeletal muscle, islet insulin and glucagon secretion, whereas a stimulatory effect is seen on hepatic gluconeogenesis.     

Immunohistology of islet amyloid polypeptide in diabetes mellitus: Semi-qantitative studies in a post-mortem series

Summary Immunoreactivity for islet amyloid polypeptide (IAPP) in the islets of Langerhans of non-insulin-dependent diabetic patients and non-diabetic patients of a non-selected post-mortem series was studied with a new polyclonal IAPP antibody. Out of 133 patients examined, 124 exhibited immunoreactivity for IAPP. Immunoreactivity was localized intra- and extracellularly and was limited to the islets of Langerhans. No extracellular immunoreactivity was observed in amyloid-negative cases. Co-localization of insulin and IAPP in the same islet-cells was verified by double staining with monoclonal insulin and polyclonal IAPP antibodies. Of 100 patients with non-insulin-dependent diabetes mellitus (NIDDM) and islet amyloid, 98 exhibited IAPP-positive deposits and 71 exhibited intracellular immunoreactivity. Evaluation of intracellular immunoreactivity and degree of islet amyloid deposition in cases of overt NIDDM revealed an inverse relationship, in that intracellular IAPP immunoreactivity were reduced in patients with developing islet amyloid deposition. Our data are consistent with the hypothesis of primary-cell dysfunction leading to amyloid formation, with subsequent disturbance of-cell homeostasis.Supported by the Johanna and Fritz Buch-Gedächtnisstiftung (Hamburg, FRG)     

Islet amyloid polypeptide, islet amyloid, and diabetes mellitus

Islet amyloid polypeptide (IAPP, or amylin) is one of the major secretory products of β-cells of the pancreatic islets of Langerhans. It is a regulatory peptide with putative function both locally in the islets, where it inhibits insulin and glucagon secretion, and at distant targets. It has binding sites in the brain, possibly contributing also to satiety regulation and inhibits gastric emptying. Effects on several other organs have also been described. IAPP was discovered through its ability to aggregate into pancreatic islet amyloid deposits, which are seen particularly in association with type 2 diabetes in humans and with diabetes in a few other mammalian species, especially monkeys and cats. Aggregated IAPP has cytotoxic properties and is believed to be of critical importance for the loss of β-cells in type 2 diabetes and also in pancreatic islets transplanted into individuals with type 1 diabetes. This review deals both with physiological aspects of IAPP and with the pathophysiological role of aggregated forms of IAPP, including mechanisms whereby human IAPP forms toxic aggregates and amyloid fibrils.     

High prevalence of pancreatic islet amyloid in patients with end-stage renal failure on dialysis treatment

Islet amyloid polypeptide (IAPP) is the main proteinaceous component of pancreatic islet amyloid, which is a characteristic feature of type 2 diabetes. The factors responsible for amyloid deposition are unclear. Patients with end-stage renal failure (ESRF) on dialysis treatment have increased insulin resistance which is associated with hypersecretion of beta-cell products. Furthermore, elevated concentrations of circulating IAPP are found in these patients due to reduced renal clearance of IAPP. To determine the prevalence of islet amyloid in this group of patients, pancreas was examined from 23 non-diabetic [aged 62 (29-79) years, median and range] and four type 2 diabetic [aged 67 (56-72) years] patients with ESRF on dialysis treatment. Pancreatic specimens from 30 non-diabetic control subjects [aged 67.5 (56-86) years] and 14 type 2 diabetic subjects without renal disease [aged 69 (48-86) years] were used as control groups. Islet amyloid was present in all type 2 diabetic patients with ESRF and in 12 out of 14 type 2 diabetic control subjects (86 per cent). Amyloid deposits were found in 8 out of 23 non-diabetic patients with ESRF (35 per cent), which was a higher prevalence than that found in non-diabetic control subjects (3 per cent) (P<0.01). This may be related to undiagnosed (pre)diabetes. Elevated secretion rates of IAPP due to insulin resistance and high circulation IAPP concentrations as a result of severely reduced renal clearance of IAPP will cause high pericellular concentrations of IAPP. This condition is likely to enhance amyloid fibril formation in pancreatic islets similar to that observed in type 2 diabetes.     

Immunochemical investigation of insulinomas for islet amyloid polypeptide and insulin: evidence for differential synthesis and storage

An affinity purified antibody to fragment 14-29 of islet amyloid polypeptide (IAPP) has been prepared. This antibody, which does not cross-react with the related molecule calcitonin gene-related peptide, was used to investigate immunochemically the presence of IAPP in normal and neoplastic human pancreatic endocrine tissue. The pattern of IAPP staining in normal pancreas mirrors that of insulin, although slight differences were observed. In neoplastic tissue, IAPP was found in 16 out of 19 tumours that were positive for insulin, and was absent from one tumour negative for insulin. In some cases there were differences in the staining patterns of IAPP and insulin. These results suggest that the synthesis and secretion of IAPP and insulin are not inter-dependent and support the concept that IAPP has a discrete biological function. Islet amyloid polypeptide was found in six out of six insulinoma amyloid deposits, suggesting that the peptide is an invariable component of these deposits. Over-expression of IAPP, with aberrant processing and/or secretion, may be the causative factor for amyloid deposition in insulinomas and in the islets of type 2 (non-insulin dependent) diabetic patients. Investigation of patients with insulinomas and of insulin cells in culture and tissue sections may help to clarify the biological function of IAPP.     

APOLIPOPROTEIN E IS ASSOCIATED WITH ISLET AMYLOID AND OTHER AMYLOIDOSES: IMPLICATIONS FOR ALZHEIMER'S DISEASE

Apolipoprotein E (ApoE) has recently been proposed as an aetiological factor of Alzheimer's disease (AD): ApoE is co-localized to amyloid plaques and neurofibrillary tangles in the brain and binds to Aβ -protein in vitro. An association of ApoE ϵ4 allele with the development of AD has been reported. Islet amyloid is formed from islet amyloid polypeptide (IAPP) in pancreatic islets of 90 per cent of patients with non-insulin-dependent diabetes mellitus (NIDDM) which, like AD, is an age-dependent pathology. The relationship of ApoE to islet amyloid and other amyloidoses is largely unknown. In this study, ApoE was localized by immunocytochemistry on pancreatic specimens from non-diabetic man, monkey, and mouse, and on amyloid-containing human tissues from pancreas, heart, brain, and intestine. All types of amyloid deposits, irrespective of the constituent peptide, site of deposition, or species, showed immunoreactivity for ApoE (ApoE-IR). Quantitative morphometry showed that similar proportions of islet amyloid were labelled for IAPP and ApoE in monkey islets. ApoE-IR was observed in pancreatic islet cells of non-diabetics. These results suggest that the association of ApoE with amyloid is non-specific for AD or to the component peptide of amyloid fibrils. If ApoE promotes amyloid formation, its synthesis in pancreatic islets could be important for the initiation or the development of pancreatic amyloid in NIDDM.     

Immunolocalization of islet amyloid polypeptide (IAPP) in pancreatic beta cells by means of peroxidase-antiperoxidase (PAP) and protein A-gold techniques.

A novel putative polypeptide hormone identified as islet amyloid polypeptide (IAPP) was recently purified from islet amyloid (IA) of diabetic humans and cats, and also from amyloid of a human insulinoma. Although the function of IAPP is yet unknown, its occurrence in pancreatic endocrine tissue and its partial amino acid sequence identity with calcitonin gene-related peptide (CGRP) suggests an endocrine regulatory effect. In the present investigation, the authors utilized antisera to insulin, glucagon, somatostatin, pancreatic polypeptide, synthetic human CGRP, and a synthetic human IAPP (7-17) undecapeptide to immunohistochemically (PAP technique) document the presence of IAPP immunoreactive cells in the islets of the cat, dog, mouse, and rat, but not in the islets of the horse or calf. In serial sections of islets from these species it was shown that IAPP immunoreactivity occurred in insulin-reactive beta cells. This observation was confirmed immunocytochemically in cat islets by means of protein A-gold probes. With protein A-gold labeling techniques, IAPP immunoreactivity was localized to the outer lucent compartment of the beta cell secretory granule, whereas insulin immunoreactivity was associated with the electron-dense core. These findings provide strong evidence that IAPP or an IAPP precursor is synthesized by beta cells and is stored in beta cell granules for subsequent co-secretion with insulin. The conservation of IAPP in humans and multiple animal species and the localization of IAPP to pancreatic beta cells provide further evidence that IAPP has an important endocrine regulatory function. The propensity of IAPP to polymerize and form IA fibrils in diabetes associated with aging may indicate that IAPP is in some way also linked to the development of Type 2 diabetes.     

A feline model of experimentally induced islet amyloidosis

The study of the pathogenesis of islet amyloidosis and its relationship to the development and progression of type 2 diabetes mellitus has been hampered by the lack of an experimentally inducible animal model. The domestic cat, by virtue of the fact that it is one of the few species that spontaneously develop a form of diabetes mellitus that closely resembles human type 2 diabetes, including the formation of amyloid deposits derived from islet amyloid polypeptide (IAPP), was considered to be an excellent candidate species in which to attempt to develop a nontransgenic animal model for this disease process. To develop the model, 8 healthy domestic cats were given a 50% pancreatectomy, which was followed by treatment with growth hormone and dexamethasone. Once a stable diabetic state was established, cats were randomly assigned to groups treated with either glipizide or insulin at doses appropriate to control hyperglycemia. Cats were maintained on this treatment regimen for 18 months and then euthanized. Based on light microscopic examination of Congo red-stained sections of pancreas, all cats were negative for the presence of islet amyloid at the time of pancreatectomy. At the end of the study all 4 glipizide-treated cats had islet amyloid deposits, whereas only 1 of 4 insulin-treated cats had detectable amyloid. In addition, the glipizide treated cats had threefold higher basal and fivefold higher glucose-stimulated plasma IAPP concentrations than insulin-treated cats, suggesting an association between elevated IAPP secretion and islet amyloidosis. Blood-glycosylated hemoglobin concentrations were not significantly different between the two treatment groups. This study documents for the first time an inducible model of islet amyloidosis in a nontransgenic animal.     

Electron-microscopical immunocytochemical study of a pancreatic islet amyloid polypeptidoma

A 50-year-old man developed a pancreatic islet tumor with liver metastases. High levels of islet amyloid polypeptide (IAPP) were recorded in plasma—35,000 pmol/L—concomitant with the occurrence of type II diabetes mellitus (the clinical syndrome has recently been described in detail) [25]. Light microscopically, the tumor contained considerable amounts of amyloid and displayed IAPP immunoreactivity both in the tumor cells and in the amyloid stroma. Electron-microscopical examination of the liver metastases showed presence of round secretory granules in the tumor cells. The granules were immunoreactive to chromogranin A and B and IAPP but not to insulin. The amyloid deposits were mainly accumulated in the extracellular spaces but were also present in the tumor cell cytoplasm. The intracellufar amyloid fibrils were, as revealed by immunogold labeling, IAPP immunoreactive and seemed to emerge from the secretory granules in the shape of radiating threads. The results show that in this particular case, the amyloid formation started already at the intracellular level and in close proximity to the lAPP-storing secretory granules. The findings may have some significance for understanding the development of pancreatic islet B-cell-related amyloidosis in type II diabetes mellitus.     

Islet amyloid polypeptide: mechanisms of amyloidogenesis in the pancreatic islets and potential roles in diabetes mellitus.

Amyloid deposits characteristically associated with pancreatic islets of those species (e.g., humans, cats, and monkeys) that develop age-associated forms of diabetes have been shown to represent a concentrated and polymerized form of a previously unknown islet-derived protein identified either as IAPP or amylin. IAPP, a highly conserved and carboxy-terminally amidated 37 amino acid polypeptide with approximately 45% amino acid sequence identity to CGRP, is produced by islet beta cells and is cosecreted with insulin in response to glucose and other secretagogues. Prepro-IAPP is synthesized in beta cells as an 89 to 93 amino acid molecule, and mature IAPP appears to be formed by enzymatic processing similar to that involved in the formation of insulin. Glucose-stimulated IAPP secretion generally parallels that of insulin and, on a molar basis, IAPP represents about 1% of the amount of insulin secreted. A significant dissociation of IAPP and insulin secretion (associated with relatively greater upregulation of IAPP secretion) is observed in response to marked hyperglycemia, suggesting that IAPP and insulin expression are differentially regulated. The amyloidogenicity of IAPP in only a very limited number of species is importantly related to the amino acid residues inherently found in the 20-29 region of IAPP from those species. The 25-28 region of human and cat IAPP is identical in structure and appears to be the most important amyloidogenic sequence common to the human and cat. In vitro fibrillogenesis studies have shown that amino acid substitutions in this region especially affect the amyloidogenicity of IAPP. Studies in dogs and cats suggest that aberrations in beta cell synthesis (or processing) of IAPP may lead to an increased concentration of IAPP in the local milieu, thus providing a second prerequisite for the self aggregation of IAPP to form islet amyloid. IAPP has been implicated to have physiological roles in glucose regulation, hemodynamics, calcium homeostasis, and as an anorectic agent. The major current interest in IAPP concerns its potential relationships to glucose metabolism and the development of type 2 diabetes. Evidence has been provided which indicates that IAPP can inhibit glucose-stimulated insulin secretion by beta cells, and that IAPP can also potentially contribute to the pathogenesis of type 2 diabetes by increasing hepatic glucose output and by inducing peripheral insulin resistance.(ABSTRACT TRUNCATED AT 400 WORDS)     

Beta amyloid and hyperphosphorylated tau deposits in the pancreas in type 2 diabetes

Strong epidemiologic evidence suggests an association between Alzheimer disease (AD) and type 2 diabetes. To determine if amyloid beta (Abeta) and hyperphosphorylated tau occurs in type 2 diabetes, pancreas tissues from 21 autopsy cases (10 type 2 diabetes and 11 controls) were analyzed. APP and tau mRNAs were identified in human pancreas and in cultured insulinoma beta cells (INS-1) by RT-PCR. Prominent APP and tau bands were detected by Western blotting in pancreatic extracts. Aggregated Abeta, hyperphosphorylated tau, ubiquitin, apolipoprotein E, apolipoprotein(a), IB1/JIP-1 and JNK1 were detected in Langerhans islets in type 2 diabetic patients. Abeta was co-localized with amylin in islet amyloid deposits. In situ beta sheet formation of islet amyloid deposits was shown by infrared microspectroscopy (SIRMS). LPS increased APP in non-neuronal cells as well. We conclude that Abeta deposits and hyperphosphorylated tau are also associated with type 2 diabetes, highlighting common pathogenetic features in neurodegenerative disorders, including AD and type 2 diabetes and suggesting that Abeta deposits and hyperphosphorylated tau may also occur in other organs than the brain.     

Islet amyloid in type 2 human diabetes mellitus and adult diabetic cats contains a novel putative polypeptide hormone.

Amyloid deposition is a very typical alteration in the islets of Langerhans in human Type 2 (non-insulin-dependent) diabetes mellitus and in feline diabetes mellitus. Amyloid infiltration is also commonly found in insulin-producing pancreatic tumors. It was shown recently that amyloid purified from an insulinoma was composed mainly of a novel polypeptide (insulinoma amyloid polypeptide, IAPP), which had partial identity with the neuropeptide calcitonin gene-related peptide (CGRP). Cat islet amyloid contained a similar polypeptide. This finding is verified in the present study, and it is shown that the cat IAPP differs from the human peptide only in two of the 16 elucidated amino acid residues. The authors now also show by N-terminal amino acid sequence analysis that human islet amyloid is of IAPP origin. Although the significance of IAPP is unknown, its occurrence in pancreatic endocrine tissue and partial identity with a known neuropeptide suggests an endocrine regulatory function.     

Naturally occurring and experimental diabetes in cynomolgus monkeys: a comparison of carbohydrate and lipid metabolism and islet pathology

Diabetes is a major health problem of increasing incidence in the United States. Diabetes research has been limited by lack of availability of good animal models, particularly for the study of comorbidities associated with diabetes. We investigated the use of cynomolgus monkeys as an animal model of both type 1 and type 2 diabetes and compared these naturally occurring diseases with streptozotocin-induced diabetes. Both type 1 diabetics and streptozotocin-induced diabetics present with sudden onset of hyperglycemia and are ketosis prone without exogenous insulin. Type 2 diabetics can have a very long period of moderate hyperglycemia and hypertriglyceridemia and only require exogenous insulin therapy if pancreatic islet reserves are depleted. Type 2 diabetes is preceded by a relatively long period of insulin resistance that is associated with obesity and dyslipidemia. As insulin resistance progresses, islet size and insulin content increases initially. However, with sustained periods of insulin resistance, islet amyloid polypeptide (IAPP) is deposited in islets and can replace normal islet architecture, resulting in an insulin-deficient state. Appearance of IAPP also occurs in human type 2 diabetics but not in conventional rodent models. Unlike type 2 diabetes, neither type 1 nor streptozotocin-induced diabetes is associated with IAPP. Rather, islets can appear normal histologically, but have decreased insulin secretion and immunostaining. Further, the amount of insulin present in the islet is correlated with plasma insulin levels following glucose challenge. Studies are ongoing to determine the pathogenic changes associated with the progression of diabetes and to find novel drug treatments for diabetics.     

Failure of islet amyloid polypeptide to inhibit basal and glucose-stimulated insulin secretion in model experiments in mice and rats

Islet amyloid polypeptide (IAPP), also known as amylin, has previously been demonstrated to occur in amyloid deposits in pancreatic islets in type 2 diabetics, and, therefore, the peptide has been suggested to be involved in the pathogenesis of diabetes. The 37 amino acid peptide shows approximately 50% homology with the intrapancreatic neuropeptide calcitonin gene-related peptide (CGRP), a peptide that inhibits insulin secretion. We therefore examined, in model experiments in mice and rats, if IAPP also exerts this effect. IAPP was given intravenously, at dose levels at which CGRP previously has been shown to inhibit insulin secretion. Thus, in mice, IAPP was injected at 0.85 and 4.25 nmol kg-1, and in rats IAPP was infused at 17 or 68 pmol min-1. However, neither basal nor glucose-stimulated insulin release was inhibited by IAPP under these experimental conditions. We also investigated if IAPP (10(-11) to 10(-6) M), when incubated in vitro with isolated, overnight-cultured rat islets, could affect insulin secretion induced by glucose (3.3, 8.3 or 11.7 mM). However, also in vitro no effect by IAPP on insulin release was observed. Hence, in mice and rats, IAPP does not inhibit insulin secretion under experimental conditions identical to those previously used to demonstrate an inhibition by CGRP. Therefore, we conclude (1) that the homologous amino acid sequence within IAPP and CGRP does not seem to be sufficient for inducing inhibition of insulin release in mice and rats and (2) that the possible involvement of IAPP in the pathogenesis of diabetes type 2 still remains speculative.     

Amyloidosis

Amyloidosis is a heterogeneous group of disorders characterized by extracellular deposition of abnormal protein fibrils which are dérived from different proteins in different forms of the disease. Asymptomatic amyloid deposition in a variety of tissues is a universal accompaniment of ageing, and clinical amyloidosis is not rare. Intracerebral and cerebrovascular β-protein amyloid deposits are a hallmark of the pathology of both sporadic and familial Alzheimer's disease, β₂-microglobulin-derived amyloid is a common complication of long term haemodialysis, and islet amyloid polypeptide is the fibril protein in the universal islet amyloidosis of type II diabetes mellitus. New fibril proteins have lately been identified in hereditary amyloidosis, including variants of gelsolin, apolipoprotein AI, lysozyme and fibrinogen. The development of radiolabelled serum amyloid P component (SAP) scintigraphy has allowed amyloid to be diagnosed non-invasively in vivo for the first time, provided unique insight into the distribution and size of amyloid deposits, and yielded novel information on the natural history and the effects of treatment. Amyloid deposits are in a state of dynamic turnover and can regress if new fibril formation is halted. The recent elucidation of the three dimensional structure of human SAP may enable the design of specific therapeutic agents.     

Islet amyloid, islet-amyloid polypeptide, and diabetes mellitus

Islet-amyloid deposits, which are a common feature of Type II diabetes mellitus, are derived from the polymerization of a putative hormone identified as IAPP. IAPP is synthesized by normal islet beta cells and probably is cosecreted with insulin. Although the physiologic function of IAPP and its role in the pathogenesis of Type II diabetes mellitus are just beginning to be unraveled, IAPP may play an important part in the development of this most common form of diabetes mellitus by opposing the action of insulin in peripheral tissues. The polymerization of IAPP to form extracellular islet-amyloid deposits may further contribute to the development of Type II diabetes mellitus by destroying islet cells and by disrupting the passage of glucose and hormones to and from them. Substantial evidence indicates that the propensity of IAPP to polymerize and form extracellular amyloid deposits in only certain species (e.g., humans, cats, and raccoons) is directly associated with an intrinsically amyloidogenic part of the molecule--i.e., positions 20 through 29 of IAPP. The inherent amyloidogenicity of IAPP in these species may be further facilitated by increased beta-cell production of IAPP, leading to a high local concentration that predisposes to polymerization. The latter possibility is supported by studies demonstrating that IAPP production by islet beta cells is increased in normoglycemic cats with impaired glucose tolerance. Although increased production of IAPP may initially cause insulin resistance, prolonged overproduction of IAPP may ultimately impair insulin secretion by leading to the progressive deposition of insoluble islet amyloid, a finding apparent in most subjects with overt diabetes. If, as these studies suggest, increased IAPP production is linked to the development of Type II diabetes mellitus, further studies must address the genetic and nongenetic factors that influence this important biologic change in humans and some animal species.     

Feline insular amyloid. Ultrastructural evidence for intracellular formation by nonendocrine cells

The objective of this ultrastructural study in cats was to investigate the early relationship of pancreatic islet cells with amyloid deposits. We used pancreatic islets from six domestic cats with minimal and apparently early amyloid deposits. Although amyloid deposits were occasionally arranged perpendicularly to beta cells, and rarely within deep invaginations of these cells, there was no consistent or convincing relationship of extracellular fibrils to any of the major islet stricted to, nongranulated perivascular cells in islets from two of the cats. Small and relatively electron-dense amyloid inclusions contained compact arrays of parallel fibrils. Larger inclusions were more electron-lucent and had loosely and randomly arranged fibrils. Indirect evidence strongly suggested that the fibril-laden inclusions resulted from intracellular production rather than from phagocytosis. The definitive identity of these amyloid-containing cells was not determined. However, these calls lacked secretory granules specific for known islet endocrine cell types and were topographically always located in close proximity to capillaries. The results of our study, therefore, do not directly support a morphologic association of amyloid fibril formation with typical islet endocrine cells. Our results do, however, draw attention to the possibility that nonendocrine cells play a key role in the pathogenesis of insular amyloidosis in the cat.     

Intracellular amyloidogenesis by human islet amyloid polypeptide induces apoptosis in COS-1 cells

Human islet amyloid polypeptide (hIAPP) is co-secreted with insulin from pancreatic islet beta cells. This peptide spontaneously aggregates in the form of fibrils, and amyloid deposits are associated with dead or degenerating beta cells, a hallmark of noninsulin-dependent diabetes mellitus. We demonstrated that COS-1 cells transfected with vectors expressing hIAPP exhibited intracellular amyloid deposits that were associated with cell death (O'Brien, Butler, Kreutter, Kane, Eberhardt, Am J Pathol 1995, 147:609-616). To establish the mechanism of cell death, we transfected COS-1 cells with vectors expressing amyloidogenic hIAPP or nonamyloidogenic rat IAPP and mutant hIAPP constructs and assayed them for markers characteristic of apoptosis and necrosis by fluorescence-activated cell sorting analysis. Amyloidogenic hIAPP-transfected COS cells contained up to threefold more apoptotic cells present at 96 hours after transfection compared with the nonamyloidogenic vector controls. The hIAPP-induced apoptosis was negligible at 24 and 48 hours after transfection and was maximal at 96 hours which parallels the time course of amyloidogenesis. Immunohistochemical staining and confocal microscopy showed that hIAPP is localized with distinct clustering in the endoplasmic reticulum and Golgi apparatus with no discernable extracellular staining. These experiments provide direct evidence that intracellular hIAPP amyloid causes cell death by triggering apoptotic pathways.     

Glycosylphosphatidylinositol-specific phospholipase D immunoreactivity is present in islet amyloid in type 2 diabetes

Numerous apolipoproteins associate with amyloid plaques. A minor high-density lipoprotein-associated protein, glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD), has recently been described by the authors and others. Since GPI-PLD is synthesized by, and secreted from, pancreatic islet beta cells, the present study examined the hypothesis that GPI-PLD associates with islet amyloid. GPI-PLD immunoreactivity was examined in pancreatic tissues from type 2 diabetic and non-diabetic humans. GPI-PLD binding to heparan sulphate proteoglycan was determined in the absence or presence of heparan sulphate or heparin. Fibril formation from human islet amyloid polypeptide was determined in the absence or presence of GPI-PLD. In non-diabetics, GPI-PLD immunoreactivity was present and co-localized with insulin, as opposed to co-localizing with amyloid in diabetics. No immunoreactivity for apolipoprotein A-I was present in islet cells or islet amyloid. Heparan sulphate proteoglycan, which is commonly present in most amyloid, bound GPI-PLD in vitro. GPI-PLD inhibited the formation of amyloid fibrils from synthetic islet amyloid polypeptide in vitro. GPI-PLD is therefore present in islet amyloid and appears to derive from local production from islets. This localization likely derives from interaction between GPI-PLD and heparan sulphate proteoglycan. Since GPI-PLD also inhibited islet amyloid polypeptide fibril formation in vitro, it is concluded that GPI-PLD may play a role in islet amyloid formation in type 2 diabetes.